| Due to the global climate change, abnormal temperature variations have become a new challenge facing plants. The plant as a sessile organism, temperature is one of the major environmental factors that regulate its growth and development. Chilling is one of the most important abiotic stresses that affect plants growth, development, yield and geographical distribution. Therefore, in order to further improve the ability of plants to adapt to low temperature, it is particularly important to study the molecular genetic mechanisms of low temperature response of plants. The main part of this thesis focusesd on low temperature adaptation mechanisms in the model plant Arabidopsis thaliana. In the appendix parts of this thesis, the molecular mechanisms of resistant to chilling stress in maize and high temperature inhibit SNC1-mediated defense response were analyzed.In the main part of this dissertation, we studied the low temperature related genes from the perspective of reverse genetics in Arabidopsis thaliana. One of these genes, AT1G70200, we names it RBD1. Because there is no piror report on RBD1, we therefore focus on RBD1 to analyze its molecular genetic mechanisms of cold-sensitive phenotype in the present study of the first part. RBD1 is only expressed in green tissues, and has a higher expression level in the mature tissues than the new emerge tissues. Loss of RBD1 affects chloroplast function and leads to chilling sensitivity. Although rbd1 mutants show chilling sensitive phenotype, RBD1 was not a cold induced gene. Furthermore, the rbd1-1 mutant is not impaired in its capacity to induce the CBF or COR expression by 4°C and the chilling sensitivity of the mutants is likely independent of the CBF pathway. RBD1 targets to chloroplast nucleoid, which is the site of r RNA processing and ribosome assembly. By analysis of chloroplast RNAs processing, expression and proteins translation, we found rbd1 mutants show a severe 23 S r RNA processing defect under cold condition, resulting in chloroplast translation inhibition and changed transcribe level of some chloroplast RNAs. Via RNA co-immunoprecipitation(IP) assay, we found low temperature induces RBD1 associates with 23 S r RNA precursor in vivo, by which mediate the processing events of 23 S r RNA. Furthermore, we found 23 S r RNA processing and protein accumulation reduce to varying degrees in the 5 RNP mutants under cold conditions, but the most significant in rbd1 mutants. In all, the first part study finds low temperature induces RBD1 bind to 23 S r RNA, by which mediate the processing of 23 S r RNA. Loss of RBD1 leads to chilling sensitivity in Arabidopsis.In the appendix part I, we studied the low temperature related genes based on the differentiate expression analysis in maize under normal and cold condition. Dr. Guang Yang had screened 296 Maize inbred lines in Jilin province at low temperature and got 3 cold-resistant inbred lines. And then he used one cold-resistant inbred line W9816 to analyze the transcripts expression at normal and low temperature. Eventually he identified 3 cold responsive genes. One of them, Zm CLC-d was analyzed in the appendix part I. 5’ RACE was conducted to clone the full length c DNA of Zm CLC-d. Zm CLC-d was induced by several abotic stresses and exogenous application of ABA and H2O2. The Zm CLC-d-overexpressing plants showed a higher tolerance to freezing, drought and salt stresses, accumulated less MDA, H2O2 and O2-, and showed higher CAT activity under cold stress. The expression of stress induced gene was significantly induced in the Zm CLC-d-overexpressing transgenic plants under the cold condition. All together, these results revealed that Zm CLC-d was involved in stress response pathways and overexpression of Zm CLC-d in Arabidopsis enhanced its stress tolerance.In the appendix part II, the temperature-dependent autoimmune mutant snc1-1 was used as the research material, and the related genes that mediate the temperature regulation of plant immunity were isolated and identified from the view of the forward genetics. Due to spontaneous immune response, snc1-1showed dwarf phenotype at 22°C, but at 28°C when the immune response is inhibited, it shows wild type Col-0 similar growth phenotype. In order to isolate and identify temperature-mediated regulating genes related to plant immunity, snc1-1 was used as background material for mutagenesis and plant growth size was used as the initial standard to screen for the plants show dwarf phenotype and maintain higher immunity at both 22°C and 28°C. These mutants were named as int(insensitive to temperature) mutant. One putative mutant, int211snc1-1, displayed a similar dwarf phenotype at 22°C and 28°C was choose as candidate int mutant. To identify the causal mutation, we performed the next-generation sequencing for mapping and gene identification. Base on the data analysis, ABCG14, an ABC-type(ATP-binding cassette) transporter, which mediated the translocation of t Z-type Cytokinin from roots to shoots via xylem to regulate shoot growth, is a good candidate gene for INT211. Phenotype analysis of abcg14 mutants and complementation test in int211snc1-1 proved INT211 is ABCG14. By Pathogen Test and q RT-PCR analysis, we found mutation in ABCG14 abolished SNC1 mediated defense response and down-regulated SNC1 and PR1 expression level. Exogenous application of t Z-type Cytokinin not only recuses the growth defect of abcg14 and abcg14snc1-1 mutants, but also enhances plant immunity. Loss of ABCG14 function miss-regulated the normal expression of cell cycle related genes, These aberent gene expressions may cause the growth defect in abcg14 mutants. In short, loss of ABCG14 affect SNC1 mediated defense response.In conclusion, my Ph D thesis research analyzed the molecular genetic mechanisms of Arabidopsis in response to low temperature, which revealed chloroplast RNA-binding protein RBD1 promotes chilling tolerance through 23 S r RNA processing. All in all, this study lays a foundation for further understanding and improving the adaptability of plants to low/ variation temperature. |
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